Electronic cigarettes (e-cig) are aggressively marketed as viable alternatives to tobacco cigarettes. E-cig are rapidly gaining acceptance in the United States and many parts of the world, especially among children and youth. However, chemical analysis of e-cig vapor/liquid has shown that many toxicants and carcinogens present in cigarette smoke are also found in a wide range of e-cig products. Notwithstanding the presence of toxicants and carcinogens in e-cig products, the biological consequences of exposure to these contaminants have not been investigated in e-cig users. This innovative collaboration brings together experts in chemical carcinogenesis, applied biology, and clinical psychology of substance abuse and addiction who will investigate, for the first time, the biological effects of e-cig as determined by early markers of genetic, epigenetic, and transcriptomic effects linked to risk of cancer. Our central hypothesis is that e-cig aerosol causes detrimental effects on the genome, epigenome, and transcriptome in oral epithelial cells of regular e-cig users. SA1: To determine the genotoxicity of e-cig aerosol in oral epithelial cels of regular e-cig users. Using a validated non-invasive brushing technique, we will collect oral epithelial cells from regular e-cig users and non-users, and subsequently quantify the formation of DNA damage in the genome overall and in representative cancer-related genes (at the nucleotide resolution level). SA2: To identify, catalogue, and interpret genome-wide DNA methylation patterns in oral epithelial cells of regular e-cig users. We will construct the whole DNA methylome of oral epithelial cells in e-cig users as compared to non-users applying our in-house `seq'-based methylation detection assay. SA3: To establish the genome-wide histone modification profiles in oral epithelial cells of regular e-cig users. We will use ChIP-seq to globally map active- and repressive histone marks in oral epithelial cells of e-cig users as compared to non-users. SA4: To analyze the global gene expression profile in oral epithelial cells of regular e-cig users. We will use RNA-seq to construct the whole transcriptome of oral epithelial cells in e-cig users as compared to non-users. In all our four Specific Aims, we will us recursively partitioned mixture models to determine relationships between genetic- and/or epigenetic alterations, transcriptional regulation, and e-cig exposure indices (e.g., frequency of use, brand and flavor, etc.). This will enable us to identify functionally important genetic- and/o epigenetic changes that are specific to e-cig exposure and predictive of its health consequences. For validation purposes, we will design functional studies involving siRNA knockdown approach, knockout models, demethylating agents, or histone deacetylase inhibitors to test experimentally whether any of the identified genetic- and/or epigenetic alteration(s) is causally linked to oral carcinogenesis. Lastly, we will integrate our data with those of public domain databases on smokers' genomic-, epigenomic-, and transcriptomic profiles to establish the relative risks/benefits of e-cig use compared to cigarette smoking.